Abstract

In the present study, we evaluated the metabolic effects of green tea polyphenols (GTPs) in high-fat diet (HFD) fed Zucker fatty (ZF) rats, in particular the effects of GTP on skeletal muscle insulin sensitivity. Body weight, visceral fat, glucose tolerance, lipid profiles and whole-body insulin sensitivity were measured in HFD-fed ZF rats after 8-week-treatment with GTP (200 mg/kg of body weight) or saline (5 ml/kg of body weight). Zucker lean rats were studied as controls. Ex vivo insulin-mediated muscle glucose uptake was assessed. Immunoblotting was used to evaluate the expression of key insulin signalling proteins in skeletal muscle. GTP treatment attenuated weight gain (P<0.05) and visceral fat accumulation (27.6%, P<0.05), and significantly reduced fasting serum glucose (P<0.05) and insulin (P<0.01) levels. Homoeostasis model assessment of insulin resistance (HOMA-IR), a measure of insulin resistance, was lower (P<0.01) in GTP-treated animals compared with ZF controls. Moreover, insulin-stimulated glucose uptake by isolated soleus muscle was increased (P<0.05) in GTP-ZF rats compared with ZF-controls. GTP treatment attenuated the accumulation of ectopic lipids (triacyl- and diacyl-glycerols), enhanced the expression and translocation of glucose transporter-4, and decreased pSer612IRS-1 and increased pSer473Akt2 expression in skeletal muscle. These molecular changes were also associated with significantly decreased activation of the inhibitory (muscle-specific) protein kinase (PKC) isoform, PKC-θ. Taken together, the present study has shown that regular ingestion of GTP exerts a number of favourable metabolic and molecular effects in an established animal model of obesity and insulin resistance. The benefits of GTP are mediated in part by inhibiting PKC-θ and improving muscle insulin sensitivity.

Introduction

Prevalence rates of obesity and the metabolic syndrome are increasing dramatically [1], and insulin resistance, especially in skeletal muscle, leads to impaired glucose tolerance, dyslipidaemia and increased cardiovascular risk [2]. Ectopic fat deposition and lipotoxicity are key features of the metabolic syndrome phenotype [3–5], which also includes reductions in insulin-stimulated glucose uptake in skeletal muscle [6–8].

In recent years, green tea consumption has become more popular, even among Western societies, in part because polyphenolic compounds in green tea, e.g. flavonoids, have shown a variety of health benefits [9–12]. Because of limited gastrointestinal (G.I) absorption of some major polyphenolic components of green tea polyphenols (GTPs), the metabolic effects reported to date have mainly been attributed to appetite suppression and dietary fat emulsification in the G.I tract [13–16].

We recently showed that GTPs exert anti-adipogenic effects on preadipocyte proliferation and reduce triglyceride accumulation in human hepatocytes [17,18]. In the present study, we aimed to extend these in vitro findings to evaluate the effects of GTP in the high-fat diet (HFD) fed Zucker fatty (ZF) rat, which is an established rodent model for studying the molecular abnormalities associated with insulin resistance, prediabetes and obesity in humans [19].

Materials and methods

Animals and HFD

Six-week-old male obese Zucker (fa/fa) rats and their age-matched lean littermates (fa/?), supplied by Monash Animal Services (Monash University, VIC, Australia), were acclimatised in communal cages at 22°C with a 12-h light, 12-h dark cycle (lights on at 0700) at the Animal Laboratory, University of Technology Sydney (UTS). All rats had access to a standard chow diet (Gordon’s Specialty Stock Feed, Sydney, Australia) and water ad libitum for 3 days. Lean rats were maintained on chow diet throughout the study. ZF rats were fed by an HFD for 2 weeks to accelerate insulin resistance and hyperglycaemia before the start of treatment. The energy percentage composition of the HFD was 59% fat, 20% carbohydrate, and 21% protein, with equal quantities of fibre, vitamins, and minerals to the standard chow diet. Body weight and food intake were monitored three times a week.

The experimental protocol, designed to minimise pain or discomfort to the animals, was approved by the ACEC (#2009-325A) of UTS and was in accordance with the National Health and Medical Research Council of Australia Guidelines on Animal Experimentation. All experimental studies on the rats were conducted at the Animal Laboratory of UTS.

GTPs and treatment protocol

The GTPs preparation (99% of total catechins consisting of: 70.9% epigallocatechin-3-gallate (EGCG,) 1.7% epigallocatechin, 7.4% epicatechin gallate, and 19.3% epicatechin) was kindly provided by Zuyi Lushen Kangyuan Co (Zuyi, Guizhou, China). After 2 weeks of HFD feeding, ZF rats (2 groups, n=12 each group) were treated with saline (5 ml/kg of body weight, ZF-control) or GTP (200 mg/kg of body weight, ZF-GTP) by daily oral gavage for 8 weeks. Meanwhile, saline (5 ml/kg of body weight) was administrated daily by gavage to chow-fed Zucker lean rats (Lean, n=12). HFD feeding was continued in the ZF rats throughout the 8-week-treatment period with GTP or saline control.

Determination of glucose tolerance and ‘whole-body’ insulin sensitivity

An oral glucose tolerance test (OGTT) was performed in conscious rats after 8 weeks of treatment. Following an overnight fast (12 h), rats were administered glucose (2 g/kg of body weight) by oral gavage. Tail vein blood samples were collected for measurement of serum glucose and insulin concentrations at baseline, 30, 60, 90, and 120 min after glucose administration.

Whole-body insulin sensitivity was measured by the homoeostasis model assessment of insulin resistance (HOMA-IR) using the formula: [fasting serum glucose (mmol) × fasting serum insulin (mU/l)]/22.5, as described by Matthews et al. [20].

Blood biochemistry assays

Blood samples from each rat were collected and serum was separated by centrifugation for the measurement of glucose, triacylglycerol (TAG), non-esterified fatty acids (NEFAs), and total cholesterol. Serum glucose concentrations were analysed using a spectrophotometric kit (Dialab Ltd, Vienna, Austria). Serum insulin concentrations were determined with an ELISA kit (Linco Research, St. Charles, MO). TAG and NEFA concentrations were analysed using an enzymatic colorimetric kit obtained from Roche Diagnostics (Indianapolis, IN) and Wako Pure Chemical Industries (Osaka, Japan), respectively. All experimental assays were performed according to the manufacturer’s instructions.

Tissue collection and ex vivo glucose uptake into isolated soleus muscle

At the end of the experiment, animals were anaesthetised after 12 h of fasting using inhalant anaesthetic gas (isoflurane and nitrous oxide). Both hindlimb soleus muscles were surgically removed and prepared for in vitro incubation. Red quadriceps muscles and visceral fat (epididymal and peri-renal adipose tissues) were quickly excised, washed by ice-cold PBS then stored at −80°C for subsequent determination of lipid deposition and molecular assays.

To assess glucose transport activity in isolated muscles, the pair of soleus muscles (n=6, each group) were placed in vials and incubated for 1 h at 37°C in 3 ml oxygenated (95% O2–5% CO2) Krebs–Henseleit buffer (KHB) containing 0.1% bovine serum albumin (BSA), 32 mM mannitol, and 8 mM glucose with continuous gas supply (95% O2/5% CO2). Following pre-incubation, muscles were incubated under basal (BSA) or insulin-stimulated (15 mU/ml) conditions for 20 min with the addition of [3H]-2-deoxy-d-glucose (2-DG, final specific activity of 2.25 mCi/mmol, Sigma–Aldrich, St. Louis, MO) for the last 20 min. Following a wash step in the incubation media, muscles were finally frozen in liquid nitrogen and later used for measurement of 2-DG uptake using the scintillation cocktail as previously described [21].

Skeletal muscle TAG and diacylglycerol measurements

Intramuscular lipids were extracted for TAG and diacylglycerol (DAG) quantification as previously described [22,23]. In brief, approximately 100 mg of red quadriceps muscle was powdered under liquid nitrogen, then homogenised at 4°C in 0.5 ml of RIPA lysis buffer (Sigma–Aldrich, St. Louis, MO). Lipids from muscle homogenate were extracted using 2 ml of chloroform/methanol solution (2:1, vol/vol). Water (1 ml) was then added to the 1 ml extraction mixture and the chloroform layer was collected as total lipids extract. The chloroform layer solution was derived into two portions. One portion was evaporated, dried and dissolved in 0.5 ml of 1-propanol (Sigma–Aldrich, St. Louis, MO) for TAG measurement. The TAG concentration was assayed using a TAG assay kit (Roche Diagnostics, Indianapolis, IN) following the manufacturer’s instructions.

DAG separation and measurements were performed by a slightly modified method of previous studies [23,24]. In brief, the second portion of the lipids–chloroform solution was evaporated under nitrogen stream (37°C) and redissolved in 100 μl of heptane:isopropyl ether:acetic acid (60:40:3, vol/vol/vol) resolving solution. Fifty microlitres of resolving sample and DAG standard (1,2-diheptadecanoin C17:0 1,2 DAG, Sigma–Aldrich, St. Louis, MO) were spotted on silica gel plates (silica plate 60, 0.25 mm; Merck). The plates were then developed with a mix of chloroform:methanol:acetone (65:15:5). After air drying, the lipid bands were visualised by spraying with a 0.2% solution of 2′,7′-dichlorofluorescein in methanol and the DAG band was identified under UV light according to the DAG standard. DAG spots were scraped off from the plates and transferred into screw tubes. Finally, DAG samples were dissolved in hexane and analysed using a Hewlett Packard 5890 series II gas chromatograph, an Agilent J&W CP-Sil 88 capillary column (50 m × 0.25 mm inner diameter), and a flame-ionisation detector (Agilent Technologies, Santa Clara, CA). The analysis was performed in the stable detector temperature (250°C) and changing the chromatograph column temperature from 160 to 225°C at the rate of 5°C/min. According to the retention times and peak intensity of 1,2 DAG standard, skeletal muscle DAG was quantified by the chromatograph profile and expressed as micromoles per gram of tissue weight.

Immunoblot analysis for functionality of insulin signalling proteins in skeletal muscle

Protein expression of insulin signalling proteins, relative to the β-actin control, was assessed by Western blotting. In brief, the pair of soleus muscles (∼100 mg, n=6 per group) was homogenised in 2 ml of RIPA lysis buffer supplemented with protease inhibitor cocktail tablets (Sigma–Aldrich, St. Louis, MO). After homogenisation, 1 ml of crude homogenate was centrifuged at 3000×g for 10 min at 4°C then the supernatants were collected for subsequent analysis of protein concentration and for immunoblotting. To subtract the crude homogenate into cytosol and particulate fractions, 1 ml of crude homogenate was first spun at 400×g for 15 min; the supernatant was then ultracentrifuged at 105000×g (Ti 50.2, Beckman, CA) for 60 min and removed. The sediment (particulate fraction) was resuspended in 0.5 ml lysis buffer. The protein concentration of each specimen was determined by the Bradford method (Bio-Rad Laboratories, CA) to ensure equal loading.

Protein (20 μg) was separated by SDS/PAGE (4–12%) and electroblotted on to PVDF membranes. Blotted membranes were then blocked with 5% skim milk in PBS with 0.05% Tween 20 and incubated with primary antibodies, including glucose transporter-4 (GLUT4), IR substrate 1 (IRS-1), pSer612IRS-1, protein kinase B (AKT), and pSer473AKT (Cell Signaling, Technology Inc., MA, U.S.A.), PKC-β2, PKC-θ, PKC-ε, PKC-ζ (Santa Cruz Biotechnology, CA, U.S.A.) and β-actin diluted at 1:1000 for 4 h. The membranes were incubated overnight at 4°C. After three washes in PBS-Tween, the membranes were incubated with corresponding horseradish peroxidase–conjugated secondary antibody (1:10000) for 2 h. Protein expression was visualised using an enhanced chemiluminescence (ECL) system (Perce, Rockford, IL) and quantified with a Quantity One 4.6.1 software of the ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA). The result was presented as a percentage of β-actin.

Statistical analysis

All values are expressed as mean ± S.E.M. Comparisons across the three groups were analysed using one-way ANOVA followed by a Duncan’s test to determine significant differences between two groups using Prism v6 software (GraphPad Inc, San Diego, CA). The total area under the curve (AUC) was calculated using the trapezoidal rule. Differences between groups with a P-value <0.05 were considered statistically significant.

Results

Effects of GTPs on body weight, visceral fat mass, and metabolic parameters

At the start of the experiment, baseline body weights of HFD-fed ZF-Con and ZF-GTP treated rats were similar (data not shown). Over 8 weeks of treatment, weight gain in GTP-treated obese ZF rats was significantly lower than in vehicle-treated ZF-Control rats (Table 1, P<0.05 vs. ZF-Con). Furthermore, GTP administration significantly decreased visceral fat mass accumulation by 27.6% compared with the ZF-Con group (Table 1, P<0.05). Thus, GTPs demonstrated a beneficial effect on visceral obesity.

Table 1
Effects of GTP on the body weight, metabolic parameters, and insulin sensitivity
ParametersLeanZF-ConZF-GTP
Body weight (g) 354.8 ± 4.40 482 ± 16.853 417 ± 11.355 
Visceral adipose1 (g) 2.36 ± 0.57 8.96 ± 0.174 6.52 ± 0.215 
Insulin sensitivity 
  Serum Glucose (mmol/l) 4.68 ± 0.34 7.84 ± 1.503 6.69 ± 0.825 
  Serum Insulin (μU/ml) 43.2 ± 4.36 1615 ± 2083 1071 ± 1196 
  HOMA-IR index 8.29 ± 0.98 541.4 ± 78.24 321.5 ± 33.86 
Lipid profiles 
  Serum TAG (mmol/l) 1.47 ± 0.06 4.08 ± 0.164 3.19 ± 0.145 
  Serum NEFAs (mmol/l) 0.76 ± 0.04 1.29 ± 0.033 1.02 ± 0.075 
  TAG content in muscle (μmol/g) 4.83 ± 0.12 17.2 ± 0.812 8.76 ± 1.076 
  DAG content in muscle (μmol/g) 0.27 ± 0.08 1.52 ± 0.214 0.98 ± 0.175 
ParametersLeanZF-ConZF-GTP
Body weight (g) 354.8 ± 4.40 482 ± 16.853 417 ± 11.355 
Visceral adipose1 (g) 2.36 ± 0.57 8.96 ± 0.174 6.52 ± 0.215 
Insulin sensitivity 
  Serum Glucose (mmol/l) 4.68 ± 0.34 7.84 ± 1.503 6.69 ± 0.825 
  Serum Insulin (μU/ml) 43.2 ± 4.36 1615 ± 2083 1071 ± 1196 
  HOMA-IR index 8.29 ± 0.98 541.4 ± 78.24 321.5 ± 33.86 
Lipid profiles 
  Serum TAG (mmol/l) 1.47 ± 0.06 4.08 ± 0.164 3.19 ± 0.145 
  Serum NEFAs (mmol/l) 0.76 ± 0.04 1.29 ± 0.033 1.02 ± 0.075 
  TAG content in muscle (μmol/g) 4.83 ± 0.12 17.2 ± 0.812 8.76 ± 1.076 
  DAG content in muscle (μmol/g) 0.27 ± 0.08 1.52 ± 0.214 0.98 ± 0.175 

ZF rats were fed with HFD for 2 weeks, then received 8 weeks treatment with vehicle (saline, 5 ml/kg of body weight) or GTPs (200 mg/kg of body weight). Metabolic parameters are represented as means ± SME (n=12 each group) after 8 weeks.

1Sum of epididymal and peri-renal adipose tissue.

2P<0.05, ZF-Con group vs Lean

3P<0.01, ZF-Con group vs Lean

4P<0.001, ZF-Con group vs Lean

5P<0.05, ZF-GTP vs ZF-Con group

6P<0.01, ZF-GTP vs ZF-Con group

After 8 weeks of treatment, fasting blood samples were obtained from each rat to examine blood glucose, insulin and lipid profiles. As shown in Table 1, ZF rats fed on the HFD became hyperlipidaemic, hyperinsulinaemic and hyperglycaemic compared with their lean controls. GTP treatment significantly decreased fasting serum glucose (P<0.05) and insulin levels by 33.6% (P<0.01). In ZF-HFD rats, GTP treatment markedly reduced fasting serum concentrations of TAG and NEFAs by 21.8%, (P<0.05) and 20.9%, (P<0.05), respectively (Table 1). HOMA-IR, an index of insulin resistance, was 40.6% less (P<0.01) in the GTP-treated rats than in ZF-con rats, indicating improved insulin sensitivity with GTP treatment.

TAG content of red quadriceps muscle was increased by 3.56-fold in vehicle-treated obese rats compared with their lean controls (Table 1, P<0.001) and GTP treatment markedly decreased TAG accumulation in skeletal muscle (P<0.01 compared with vehicle treated group). Together with reductions in serum TAG levels, this result indicates that GTP attenuates obesity and HFD-feeding induced ectopic lipid deposition in skeletal muscle.

Intramuscular DAG content in red quadriceps muscle was higher in ZF-Con rats than the lean controls ( <0.001). Although DAG remained higher than in the lean control, DAG levels decreased by 35.5% in GTP-treated ZF rats compared with the vehicle treated ZF group (P<0.05), suggesting that GTP also attenuated the accumulation of lipid intermediates in skeletal muscle.

Effects of GTP on glucose disposal and insulin sensitivity during OGTT

The responses of plasma glucose to an oral glucose load (2 g/kg of body weight) are shown in Figure 1. Compared with the lean control rats, HFD-fed obese rats showed higher glucose levels at baseline and after glucose administration. Chronic treatment with GTP led to significantly increased glucose disposal at the respective time points of 60 and 90 min after glucose loading (Figure 1A). The total integrated AUC for the glucose-time profile during the OGTT was reduced by 19.8% (Figure 1B, P<0.05) in the GTP-treated group, indicating that GTP improved glucose tolerance in HFD-fed ZF rats. The basal levels of insulin and the total integrated AUC for the insulin-time profile were significantly lower in ZF-GTP than ZF-vehicle rats during the OGTT (Figure 1C,D). Together with the improved OGTT and HOMA-IR measurements, this result reflects a significant enhancement of whole-body insulin sensitivity in HFD-ZF rats treated with GTP.

Effects of GTP on glucose disposal and insulin secretion response to oral glucose loading

Figure 1
Effects of GTP on glucose disposal and insulin secretion response to oral glucose loading

A total of (2 g/kg of body weight) in chow fed Zucker lean and HFD-fed ZF rats treated with saline (5 ml/kg of body weight, ZF-control) or GTP (200 mg/kg of body weight, ZF-GTP). (A,C) Serum glucose and insulin levels at appropriate time points. (B,D) AUCs of glucose and insulin profiles. Data are means ± SME (n=12 each group). ***P<0.001 vs Lean and #P<0.05 vs ZF-Con group.

Figure 1
Effects of GTP on glucose disposal and insulin secretion response to oral glucose loading

A total of (2 g/kg of body weight) in chow fed Zucker lean and HFD-fed ZF rats treated with saline (5 ml/kg of body weight, ZF-control) or GTP (200 mg/kg of body weight, ZF-GTP). (A,C) Serum glucose and insulin levels at appropriate time points. (B,D) AUCs of glucose and insulin profiles. Data are means ± SME (n=12 each group). ***P<0.001 vs Lean and #P<0.05 vs ZF-Con group.

Effects of GTP on insulin-stimulated glucose uptake in isolated skeletal muscle

Insulin-stimulated glucose transport activity was assessed in isolated soleus muscle strips to determine the cellular locus for the enhancement of insulin action. Rates of 2-DG uptake in the absence of insulin were significantly lower in soleus muscles of ZF-con and ZF-GTP rats compared with the lean controls but there was no significant difference in 2-DG uptake by soleus muscles from ZF-con and ZF-GTP (Figure 2). After insulin stimulation, glucose uptake was increased in soleus muscles from lean controls, ZF-controls and GTP-treated ZF rats by 63.0, 21.3, and 57.6%, respectively. The results of the present study demonstrate skeletal muscle insulin resistance in HFD-ZF rats and show that GTP treatment improves this metabolic defect.

Effects of GTP on glucose uptake by soleus muscle

Figure 2
Effects of GTP on glucose uptake by soleus muscle

Ex vivo 2-DG uptake by soleus muscle from lean and HFD-fed ZF rats with 8 weeks of saline or GTP treatment incubated with 0.1% BSA as Basal condition or with 15 mU/ml of insulin. Data are means ± SME (n=6). **P<0.01 and ***P<0.001 vs Lean and #P<0.05 vs ZF- Con group.

Figure 2
Effects of GTP on glucose uptake by soleus muscle

Ex vivo 2-DG uptake by soleus muscle from lean and HFD-fed ZF rats with 8 weeks of saline or GTP treatment incubated with 0.1% BSA as Basal condition or with 15 mU/ml of insulin. Data are means ± SME (n=6). **P<0.01 and ***P<0.001 vs Lean and #P<0.05 vs ZF- Con group.

Effects of GTP on expression of insulin signalling proteins in skeletal muscle

To further understand the molecular mechanisms for the improved glucose disposal during OGTTs and the increased insulin-stimulated glucose uptake in skeletal muscle by GTP, immunoblot analysis was employed for detecting the expression of various key proteins involved in insulin signal transduction, e.g. GLUT4 is a critical downstream element of insulin signalling in skeletal muscle.

GLUT4 protein expression was detected in cytosol and membrane fractions of soleus muscle. Figure 3A,B shows that protein expression was significantly higher in soleus muscle of lean control and ZF-GTP rats compared with ZF controls. GLUT4 activation was determined by the translocation from cytosol to membrane fraction [25], which is presented as the membrane/cytosol ratio. Figure 3C shows that translocation of GLUT4 in soleus muscle was lower in ZF-control rats compared with lean rats (P<0.01) and that GLT4 translocation was markedly increased in ZF-GTP animals (P<0.05 vs ZF-control).

GTP treatment enhanced GLUT4 expression and translocation

Figure 3
GTP treatment enhanced GLUT4 expression and translocation

(A) Representative image of immunoblots of GLUT4 in soleus muscles isolated from lean and HFD-fed ZF rats with 8 weeks of saline or GTP treatment. (B) Quantified GLUT4 expression with ChemiDoc XRS system and presented as percentage of β-actin. (C) GLUT4 translocation is presented as the ratio of membrane over cytosol. Data are means ± SME (n=6). *P<0.05 vs Lean and #P<0.05 vs ZF-Con group.

Figure 3
GTP treatment enhanced GLUT4 expression and translocation

(A) Representative image of immunoblots of GLUT4 in soleus muscles isolated from lean and HFD-fed ZF rats with 8 weeks of saline or GTP treatment. (B) Quantified GLUT4 expression with ChemiDoc XRS system and presented as percentage of β-actin. (C) GLUT4 translocation is presented as the ratio of membrane over cytosol. Data are means ± SME (n=6). *P<0.05 vs Lean and #P<0.05 vs ZF-Con group.

Insulin facilitates glucose uptake into muscle cells by translocating GLUT4 towards the cell surface through a signalling pathway that involves the insulin receptor (IR) and IRS-1/phosphatidylinositol 3 kinase (PI3K) and Akt activation [25,26]. Figure 4 shows that serine phosphorylated proteins of IRS-1 at the Ser612 site were significantly higher in ZF rats than the lean and ZF-GTP rats (both P<0.05). On the other hand, Ser473 phosphorylation of Akt was significantly higher in the lean controls and GTP-treated ZF rats than in ZF-Con rats (both P<0.01 vs Lean control ZF-GTP rats).

Effects of GTP on phosphorylation of IRS-1 and Akt

Figure 4
Effects of GTP on phosphorylation of IRS-1 and Akt

Representative immunoblots (A,B) and quantitative analysis of phosphorylated Ser612IRS-1/IRS-1 (C) and phosphorylated Ser473Akt/Akt (D) in soleus muscle of chow-fed lean rats and HFD-fed ZF rats treated with saline or with GTP. Data are expressed as means ± SME (n=6). *P<0.05 and **P<0.01 vs Lean and #P<0.05 and ##P<0.01 vs ZF-Con group.

Figure 4
Effects of GTP on phosphorylation of IRS-1 and Akt

Representative immunoblots (A,B) and quantitative analysis of phosphorylated Ser612IRS-1/IRS-1 (C) and phosphorylated Ser473Akt/Akt (D) in soleus muscle of chow-fed lean rats and HFD-fed ZF rats treated with saline or with GTP. Data are expressed as means ± SME (n=6). *P<0.05 and **P<0.01 vs Lean and #P<0.05 and ##P<0.01 vs ZF-Con group.

Effects of GTP on the expression of protein kinase C isoforms in skeletal muscle

The protein kinase C (PKC) family of isoenzymes are important modulators of insulin signalling. Previous studies have demonstrated that PKC activation negatively affects AKT-GLUT4 activity [27,28]. We next investigated the possibility that GTP treatment improves the insulin signalling pathway in skeletal muscle through altered regulation of key PKC isoforms.

Expression of PKC-β2, -ε, -θ, and -ζ were clearly identified in both the cytosol and membrane fractions of soleus muscle (Figure 5-1 and Table 2).

Effects of GTP on expression of PKC isoforms

Figure 5-1
Effects of GTP on expression of PKC isoforms

Representative immunoblots of PKC-β2, PKC-ε, PKC-θ, PKC-ζ, and β-actin in the cytosol and membrane fraction of soleus muscle from chow-fed lean rats and HFD-fed ZF rats treated with saline or with GTP.

Figure 5-1
Effects of GTP on expression of PKC isoforms

Representative immunoblots of PKC-β2, PKC-ε, PKC-θ, PKC-ζ, and β-actin in the cytosol and membrane fraction of soleus muscle from chow-fed lean rats and HFD-fed ZF rats treated with saline or with GTP.

Table 2
PKC isoforms expression in cytosol and membrane fraction of soleus muscle
LeanZF-ConZF-GTP
CytosolMembraneCytosolMembraneCytosolMembrane
PKC-β2 76.9 ± 9.2 107.1 ± 23 61.3 ± 5.1 127.2 ± 21.8 62.3 ± 9.5 61.3 ± 5.7 
PKC-ε 55.5 ± 6.2 62.1 ± 4.6 66.2 ± 5.8 99.6 ± 7.1 77.9 ± 9.4 97.7 ± 7.9 
PKC-θ 70.6 ± 12 53.1 ± 2.4 39.7 ± 5.2 111.2 ± 10.4 104.7 ± 15.6 73.4 ± 4.8 
PKC-ζ 111.1 ± 32 86.9 ± 6.5 34.5 ± 2.9 96.3 ± 7.6 104.9 ± 13.1 164.1 ± 10.5 
LeanZF-ConZF-GTP
CytosolMembraneCytosolMembraneCytosolMembrane
PKC-β2 76.9 ± 9.2 107.1 ± 23 61.3 ± 5.1 127.2 ± 21.8 62.3 ± 9.5 61.3 ± 5.7 
PKC-ε 55.5 ± 6.2 62.1 ± 4.6 66.2 ± 5.8 99.6 ± 7.1 77.9 ± 9.4 97.7 ± 7.9 
PKC-θ 70.6 ± 12 53.1 ± 2.4 39.7 ± 5.2 111.2 ± 10.4 104.7 ± 15.6 73.4 ± 4.8 
PKC-ζ 111.1 ± 32 86.9 ± 6.5 34.5 ± 2.9 96.3 ± 7.6 104.9 ± 13.1 164.1 ± 10.5 

Western blot analysis of PKC-β2, PKC-ε, PKC-θ, and PKC-ζ in both cytosolic and membrane protein fractions of soleus muscle from the lean control and ZF rats with GTP or saline treatment. The quantified data are calculated as percentage of optical density of each brand over corresponding β-actin. Data are shown as mean ± SEM, n=6 each group.

Figure 5-2 shows that there were no significant differences in expression of PKC-β2 and -ε in cytosol and membrane fractions of skeletal muscle among all the groups (Figure 5 and Table 2).

Effects of GTP on translocation of PKC isoformsEffects of GTP on translocation of PKC isoforms

Figure 5-2
Effects of GTP on translocation of PKC isoformsEffects of GTP on translocation of PKC isoforms

The quantitative analysis of PKC-β2 (A), PKC-ε (B), PKC-θ (C), PKC-ζ (D) isoforms expression. The translocation of PKC isoforms is presented as the ratio of PKC expression in the membrane over cytosol fractions. Data are expressed as means ± SME (n=6). **P<0.01 and ***P<0.001 vs Lean and ###P<0.001 vs ZF-Con group.

Figure 5-2
Effects of GTP on translocation of PKC isoformsEffects of GTP on translocation of PKC isoforms

The quantitative analysis of PKC-β2 (A), PKC-ε (B), PKC-θ (C), PKC-ζ (D) isoforms expression. The translocation of PKC isoforms is presented as the ratio of PKC expression in the membrane over cytosol fractions. Data are expressed as means ± SME (n=6). **P<0.01 and ***P<0.001 vs Lean and ###P<0.001 vs ZF-Con group.

However, expression of PKC -θ and -ζ in the membrane fraction was significantly higher in soleus muscles of HFD-ZF control rats compared with the lean rats. In particular, PKC-θ translocation was almost completely abolished (Figure 5-2C) and PKC-ζ translocation was reduced significantly (Figure 5-2D) by GTP treatment.

Discussion

Polyphenols, either from dietary or neutraceutical supplements, have prompted considerable interest but few if any previous studies have investigated the metabolic effects of GTP in vivo using a rodent model of obesity, insulin resistance, and dyslipidaemia. A number of murine models of metabolic syndrome are established, e.g. the spontaneously hypertensive-obese rat and the Otsuka Long–Evans Tokushima fatty rats, but the Zucker obese fatty (ZF) rat has well-defined metabolic abnormalities typical of insulin resistance in humans [29]. We and others have also shown that HFD feeding exacerbates the hyperinsulinaemia and hyperlipidaemia in obese ZF rats and leads to mild hyperglycaemia [30].

In the present study, we showed that chronic treatment (8 weeks) with GTP to HFD-fed ZF rats significantly reduced weight gain, improved glucose tolerance and insulin resistance, and attenuated the lipid accumulation that often causes ectopic fat deposition and complications such as fatty liver disease.

GTP treatment reduced visceral fat accumulation and also reduced ectopic fat (TGA and DAG) deposition in skeletal muscle. Interestingly, the ex vivo study showed enhancement of glucose uptake by skeletal muscle under insulin-stimulated conditions, which is consistent with the results from the in vivo study that glucose disposal was improved in GTP-treated animals following an OGTT. Administration of GTP also improved whole body insulin sensitivity, as evidenced by lower HOMA-IR values in the treatment group.

Green tea is a popular beverage containing polyphenols. Previous studies have reported various biological and health effects of ingesting tea polyphenols, especially the active compound (-)-EGCG, for example in suppressing appetite, modifying dietary fat emulsification in the G.I tract, reducing nutrient absorption and inhibiting hepatic lipogenesis [15,18,31,32]. The results of this study extend the metabolic effects of GTP to include improvements in muscle insulin sensitivity, glucose tolerance, and favourable lipid effects that will likely reduce the risk of fatty liver disease. Our previous work showed that chronic treatment with GTP attenuated hepatic fat accumulation and normalised serum aspartate transaminase (AST) and alanine transaminase (ALT) levels in an animal model of non-alcoholic fatty liver disease (NAFLD) [18,30].

Skeletal muscle is the largest organ in the body. It plays a pivotal role in glucose homeostasis, as it can account for up to 40% of the body weight and for up to 80–90% of insulin-stimulated glucose disposal [6–8]. Loss of insulin sensitivity in skeletal muscle results in elevated postprandial blood glucose levels and hyperinsulinemia, eventually leading to pancreatic β-cell exhaustion and overt hyperglycaemia [2].

In the present study, we demonstrated that GTP-treated ZF rats showed a greater ability to facilitate glucose disposal into muscle and improved ‘whole body’ insulin sensitivity, as evidenced by the decreased AUC of glucose and reductions in hyperinsulinemia and HOMA-IR values. Insulin-stimulated glucose uptake by isolated soleus muscles from GTP-treated ZF rats was significantly higher than in the muscles from ZF-control rats, suggesting that GTP improved muscle insulin sensitivity.

It is well recognised that intramyocellular lipid accumulation is positively correlated with skeletal muscle insulin resistance [6,33]. In this study, we have identified higher muscle DAG and TAG content in HFD-fed ZF rats than in their lean controls. Furthermore, treatment with GTP significantly attenuated the lipid accumulation in red quadriceps muscles. DAG is an intermediate lipid involved in the synthesis of phospholipids and TAG. As a signalling regulator, DAG decreases the response to insulin in skeletal muscle through activation of PKC, which in turn down-regulates insulin signalling [28,34,35].

To understand the molecular mechanism by which GTP improves whole body and skeletal muscle insulin sensitivity, we measured glucose transport activity and the expression of key insulin signalling proteins in skeletal muscle. Insulin stimulates glucose uptake by inducing the translocation of GLUT4 from intracellular storage sites (cytosol) to the plasma membrane, where it facilitates the uptake of glucose into skeletal muscle to be stored as glycogen [20]. We found that GLUT4 protein expression in the cytosol fraction was similar in all groups of rats but GLUT4 protein levels were higher in the membrane fraction of soleus muscles from lean and GTP treated animals. The increased translocation of GLUT4 in GTP-treated rats provided a molecular explanation for the faster glucose disposal during an OGTT. The ex vivo studies showed that the enhanced glucose uptake by soleus muscle in GTP-treated rats only occurred after insulin stimulation, indicating that GTP improved muscle insulin sensitivity. Insulin facilitates GLUT-4 translocation via a complex phosphorylation process involving multiple signalling proteins, including the IRS proteins, PI3-kinase and Akt [25,26,36]. Insulin-stimulated tyrosine phosphorylation of IRS-1 by the IR results in intracellular transduction of the insulin signal to recruit and activate PI3K, which in turn activates Akt2 through phosphorylating at Ser474 and Thr309, respectively [37].

To obtain further insights into the mechanism of action of GTP on the insulin signalling pathway, we detected total and phosphorylated IRS1 and Akt in soleus muscle using immunoblotting. In our study, phosphorylation of IRS-1 was significantly higher at the Ser612 site in ZF control rats compared with the lean controls and pSer612IRS-1 in GPT treated rats was similar to lean controls. Interestingly, phosphorylated Akt at the Ser473 site was significantly increased in soleus muscle of GPT-Zucker rats. Thr308 and Ser473 phosphorylation is essential for AKT activity and Akt activation plays a pivotal role in the regulation of GLUT4 delivery to the cell surface and for glucose uptake into cells [38].

A recent cellular study demonstrated that EGCG (the major bioactive compound in GTP) alone or combined with rosiglitazone increased the phosphorylation of AktS473, leading to increased glucose uptake into C2C12 cells [39]. In the present in vivo study, we demonstrated reduced pSer612IRS-1, together with increased pSer473Akt2, which suggests that GTP may target skeletal muscle and exert useful insulin-sensitising effects.

Increased serine phosphorylation of IRS-1 on Ser302, Ser307, Ser612, and Ser632 has been identified in several rodent models of insulin resistance [39,40]. Furthermore, a muscle-specific triple serine to alanine mutant mouse (IRS-1 Ser → Ala302, Ser → Ala307, and Ser → Ala612) is protected from HFD-induced insulin resistance in vivo [41]. Serine phosphorylation of IRS proteins can occur in response to a number of intracellular serine kinases [30]. We have observed an increase in PKC-θ expression in the skeletal muscles of ZF rats and aged rats with insulin resistance [23,31,42], which is consistent with recent reports by Szendroedi et al. [27]. Here, we further investigated whether GTP effects are at least partly due to down-regulation of PKC isoform activation. The results in Figure 5 and Table 2 show increased translation of novel PKC-θ and atypical PKC-ζ isoforms in soleus muscle of Zucker control rats compared with the lean controls. GTP treatment partially prevented PKC-ζ translocation and completely abolished PKC-θ translocation. This finding indicates that: (i) PKC-θ plays a major role in the hyperserine phosphorylation of IRS-1, which in turn promotes insulin resistance and (ii) the underlying mechanism of the insulin-sensitising effect of GTP may involve the PKC-pathway, in particular attenuating the negative effect of PKC-θ on insulin sensitivity in skeletal muscle. The inhibitory effect of GTP on PKC activation may be secondary to reduced DAG accumulation in skeletal muscle because the novel PKC isoforms (δ, ε, θ, η) are activated only by DAG [35] and PKC-θ is a major isoform involved in skeletal muscle insulin resistance [23,27,42,43].

There are a number of important limitations of the present study which merit discussion. Firstly, G.I absorption of some polyphenolic compounds is limited and variable. It is therefore a major limitation that metabolite and other polyphenol components were not measured directly in blood. Secondly, the dose of GTP used in the present study is similar to that used in previous experimental models but without more pharmacokinetic information it is unclear whether this dose of GTP would convert into an excessive dose in humans which has been linked to hepatotoxicity [44]. The FDA has issued guidance on calculating a human equivalent dose (HED) based on the animal dose (in mg/kg) multiplied by a ratio of 0.162 [45]. Thus, the animal dose of GTP used in these studies (200 mg/kg) converts into an HED of 32 mg/kg.

In conclusion, the present study has shown that a purified extract of GTPs administered for 8 weeks to HFD-fed obese Zucker rats is associated with improved whole-body insulin sensitivity, in part by increasing GLUT-4 translocation and insulin-stimulated glucose uptake in skeletal muscle. The insulin-sensitising effects of GTP supplementation may be due to a reduction in PKC activation, which is known to negatively affect insulin signalling in multiple tissues in this rodent model of type 2 diabetes. There is uncertainty about the appropriate dose and long-term safety of GTP supplementation in humans.

Clinical perspectives

  • Obesity and type 2 diabetes are characterised by peripheral (muscle and fat) insulin resistance.

  • The present study shows that in a rodent model of obesity and HFD-induced insulin resistance GTP supplementation had favourable metabolic effects.

  • The present study highlights GTP as a potential nutraceutical supplement but the most appropriate dose and long-term safety have yet to be established in humans.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This work was supported by a Special International Collaboration Grant from the Ministry of Science and Technology of the People’s Republic of China [grant number S2011GR0387].

Author Contribution

X.Q. conceived and designed the study. J.C. (visiting scholar under X.Q.’s supervision) and Y.T. conducted majority of animal experiments and laboratory assays. J.Z. and L.X. contributed to chemical analysis of GTP. M.J. contributed to analysis of the immunoblotting imaging bands. Y.T. and J.Z. conducted statistical analysis for all data collected and prepared figures and tables for the manuscript. X.Q. wrote the manuscript and M.J. reviewed and revised the final draft of the manuscript.

Acknowledgements

We are grateful to Zuyi Lushen Kangyuan Co (Meitan, Guizhou, China) for supporting the present study by providing GTPs. The authors would also like to thank Dr Yali Sun’s advice for chemical analysis of GTPs.

Abbreviations

     
  • 2-DG

    2-deoxy-d-glucose

  •  
  • AKT

    protein kinase B

  •  
  • AUC

    area under the curve

  •  
  • BSA

    bovine serum albumin

  •  
  • DAG

    diacylglycerol

  •  
  • EGCG

    epigallocatechin-3-gallate

  •  
  • GLUT4

    glucose transporter-4

  •  
  • GTP

    green tea polyphenol

  •  
  • G.I

    gastrointestinal

  •  
  • HED

    human equivalent dose

  •  
  • HFD

    high-fat diet

  •  
  • HOMA-IR

    homoeostasis model assessment of insulin resistance

  •  
  • IR

    insulin receptor

  •  
  • IRS-1

    IR substrate 1

  •  
  • NEFA

    non-esterified fatty acid

  •  
  • OGTT

    oral glucose tolerance test

  •  
  • PI3K

    phosphatidylinositol 3 kinase

  •  
  • PKC

    protein kinase C

  •  
  • PKC-β2

    protein kinase C-β2

  •  
  • PKC-ε

    protein kinase C-ε

  •  
  • PKC-ζ

    protein kinase C-ζ

  •  
  • PKC-θ

    protein kinase C-θ

  •  
  • TAG

    triacylglycerol

  •  
  • UTS

    University of Technology Sydney

  •  
  • ZF rat

    Zucker fatty rat

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